Establishment of a Monoclonal Antibody Against chGILZ
Establishment of a Monoclonal Antibody Against chGILZ
复制标题
抗chGILZ单克隆抗体的建立
DOI：
10.1089/mab.2017.0006
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发表时间：
2017
期刊：
影响因子：
--
通讯作者：
Yongqiang Wang
中科院分区：
文献类型：
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作者：
Zhiyuan He;Yongqiang Wang
鸡糖皮质激素诱导的亮氨酸拉链（chGILZ）参与禽类免疫抑制性疾病的炎症反应。本研究旨在建立抗chGILZ的单克隆抗体（MAb），并探讨其在临床诊断中的分布。将从鸡胚成纤维细胞（CEF）中克隆的基因分别克隆到原核表达载体pET-28 a和pGEX-6p-1中。重组表达载体转化大肠杆菌BL 21（DE 3）。成功表达并纯化了重组蛋白His-chGILZ和GST-chGILZ。通过将小鼠骨髓瘤细胞系SP 2/0与免疫小鼠的脾细胞融合来制备杂交瘤，并用纯化的GST-chGILZ进行筛选。两个杂交瘤（1D 3和3F 4）是有效的检测重组和天然chGILZ蛋白，并进行了分离和表征。Westernblot检测表明，单克隆抗体不与人GILZ蛋白发生反应，但能识别chGILZ蛋白。这些数据和试剂将有助于阐明鸡传染性法氏囊病等免疫抑制性疾病的分子机制
The chicken glucocorticoid-induced leucine zipper (chGILZ) participates in the inflammation of avian immunosuppressive diseases. We aimed to establish a monoclonal antibody (MAb) against chGILZ and to investigate its distribution in clinical diagnosis. A gene cloned from chicken embryo fibroblast (CEF) cell was inserted into the expression vector pET-28a and pGEX-6p-1. The recombinant expression vectors were transformed into Escherichia coli BL21 (DE3). Then the recombinant proteins His-chGILZ and GST-chGILZ were successfully expressed and purified. The hybridomas were developed by fusing mouse myeloma cell line SP2/0 with splenocytes of immunized mice and screened with purified GST-chGILZ. Two hybridomas (1D3 and 3F4) were effective in detecting both recombinant and native chGILZ proteins and were isolated and characterized. The MAbs did not react with human GILZ protein, but could recognize chGILZ by Western blot assay. These data and reagents will be of great assistance to elucidate the molecular mechanism of avian immunosuppressive diseases, such as the infectious bursal disease